Cyanobacteria, also called blue-green algae, are photosynthetic organisms that use chlorophyll A and water to reduce carbon dioxide and generate energy-containing compounds. Spirulina are free-floating, filamentous cyanobacteria that include the species Arthrospira platensis and Arthrospira maxima. These two species were formerly classified in the genius Spirulina, but are now classified in the genus Arthrospira. However, the term “Spirulina” remains in use.
Cyanobacteria are generally amenable to genetic manipulation. However, genetic engineering tools for Spirulina are limited. Many techniques for genetic manipulation are based on introducing exogenous (foreign) genetic material into a bacterial cell. Cells must be in a state of “competence” to take up genetic material from the surrounding environment. Some types of bacteria are able to naturally take up genetic material. These types of bacteria are referred to as having “natural competence.” More commonly, “artificial competence” is induced by making a cell temporarily permeable to genetic material. Techniques for introducing artificial competence by increasing the permeability of an outer cell membrane include incubation in chemical solutions, heat shock, and electroporation which subjects a cell to an electric field. A cell in a state of competence that uptakes exogenous genetic material and incorporates the new genetic material into its genome is said to be have undergone “transformation.”
Spirulina has been long recognized as difficult to transform by random integration of DNA into a Spirulina chromosome, and applicant is not aware of any reports claiming modification of the Spirulina genome by targeted introduction of DNA into specific, predetermined chromosome locations. Attempts using electroporation to introduce a gene for chloramphenicol resistance have resulted in chloramphenicol resistance under certain electroporation conditions, but the transformation was not stable (i.e. the chloramphenicol resistance could not be sustained). Subsequent attempts to transform Spirulina with a gene for chloramphenicol resistance coupled to a strong promoter by using electroporation achieved cells that grew in the presence of chloramphenicol for 12 months, but the method only allowed the gene for chloramphenicol resistance to be located at random (un-targeted) locations in a Spirulina chromosome, and even this random integration of exogenous DNA was not conclusively demonstrated. Recently, random mutagenesis has been achieved in S. platensis (A. platensis) using atmospheric and room-temperature plasma (ARTP). However, random mutagenesis does not transform the Spirulina cells through introducing exogenous genetic material, rather mutations are introduced at random sites in the genome. A lack of understanding of how to stably introduce foreign DNA to predetermined chromosome locations into cyanobacteria, Spirulina in particular, is recognized as a challenge to working with cyanobacteria as compared to other organisms such as E. coli or yeast. Although there was some level of success with random mutagenesis, this research also highlighted the continued lack of an effective system for mutation of S. plantensis by introduction and expression an exogenous gene. Moreover, none of the techniques described above have attempted to introduce targeted mutations to specific, pre-determined regions of the Spirulina genome.
A need still exists for a technique to efficiently create stable transformants in Spirulina. Moreover, there is also a need for techniques that allow for targeted introduction of mutations in the Spirulina genome.